DESCRIPTION (provided by applicant : ABS Francisella tularensis (Ft) is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia, a disease associated with high morbidity and mortality in humans. Pneumonic tularemia, caused by inhalation of aerosolized Ft or hematogenous spread of Ft from distant infectious foci to the lung, is the most lethal form of the disease, resulting in up to 50% mortality without treatment. Characterized by profound disruption of the alveolar epithelium and profuse alveolar and interstitial mononuclear infiltrates, Ft pneumonia is an acute inflammatory infection often resulting in lung damage. Alveolar macrophages are the primary cell type targeted by Ft following infection, serving as both a site of bacterial replication and vehicle for subsequent dissemination. However, Ft encounters other cell types following inhalation infection, including lung epithelial cells. Recently, Ft interaction with alveolar type II and type I (ATII and ATI) pneumocytes was reported during early stages of murine pneumonic tularemia. The significance of these interactions in the lifecycle of Ft remains unclear, and no studies to date have addressed the importance of Ft interactions with human alveolar pneumocytes. Preliminary studies described in this proposal indicate that Ft infects, replicates, and persists in the human ATII-like cell lin A549 without inducing host cell cytopathic effects even at high multiplicities of infection, a phenotype that contrasts that seen following infection of Ft in other cell types including macrophages where host cytopathic effects are observed within 24 hrs. This proposal seeks to fill our current knowledge gap by investigating aspects of Ft interactions with primary human ATII and ATI pneumocytes. We hypothesize that ATII and/or ATI pneumocytes may serve as an intracellular reservoir for virulent Ft within the human lung, allowing a subset of bacteria to be maintained within a protected niche where they are able to modulate the subsequent host response. To address this hypothesis, three specific aims have been proposed. First, a primary human ATII/ATI infection model for Ft will be established and used to characterize internalization, replication, and trafficking of the bacterium. Second, bacterial determinants expressed by Ft within lung pneumocytes will be identified using RNA-seq technology and by screening a >20,000-member Schu S4::himar1 transposon mutant library in A549 cells for defects in cell cytotoxicity suppression. Finally, a subset of pneumocyte-expressed genes identified above will be targeted for disruption and characterization using various in vitro and in vivo model systems of infection. Collectively, these studies are expected to provide novel insights into the importance of ATII/ATI pneumocytes in Ft pathogenesis. This information may lead to identification of novel therapeutic targets or vaccine candidates to combat Ft infection.